The CNS of adult mammals, as compared to the peripheral nervous system or nervous system of other organisms, has extremely limited capacity for axon regeneration and synapse reformation. Specific factors limiting adult mammalian regeneration of axons and synapses have been identified, but they provide an incomplete explanation for poor adult mammalian CNS regeneration. We propose the first genome-wide shRNA-based screen for endogenous genes limiting the repair of axons and synapses in the mammalian CNS. In the first R21 Phase of this project we will develop a tissue culture screening method for axonal and synaptic regeneration relevant to cortical neurons. In Preliminary Studies, we conducted a pilot loss-of-function screen with 1087 shRNAs targeting mouse phosphatases, monitoring total regenerative axon length. We will confirm activity and assess their interaction with previously identified extrinsic inhibitors of axonal regeneration. We also propose to extend our preliminary analysis of the phosphatome in regeneration by imaging growth cones and categorizing their morphology. In addition, we will assess the reformation of synapses during regeneration, and demonstrate its utility in the phosphatome screen of regeneration. Finally, we will demonstrate that in vivo axonal regeneration is regulated by at least one novel phosphatase identified in the pilot screen. After accomplishing these milestones, we will screen the mouse genome for effects on CNS axonal and synapse regeneration during the R33 Phase. The cortical scrape assay will be probed at the genome-wide level with lentiviral shRNAs. Metrics for axon regeneration, growth cone morphology and synapse reformation from the R21 phase will be employed. The focus will be to identify all genes that naturally limit corticl axon and synapse regeneration, by demonstrating enhanced regeneration after knockdown of individual genes. We will determine if newly discovered endogenous brakes on regeneration are dominant with respect to the action of extrinsic inhibitors. Together, these Aims will provide the first functional genomic assessment of axonal and synaptic regeneration in the adult mammalian central nervous system. The findings will have with high relevance for the development of novel therapeutics for neurological disorders.